The present invention relates to a method of separating blood components. More specifically, it relates to a method of obtaining functionally and morphologically intact leucocytes and thrombocytes.
Leucocytes (white blood cells) are trace cell components of the blood. They perform various functions in the body, such as the defense against infections, the degradation of destroyed biological material (phagocytosis), and the production of materials regulating cell growth and natural resistance such as hormones, immunological mediators and immunoglobulins, prostaglandins, and histamine. Undesirable or impaired functions of the leucocytes are manifest in allergy, tumor survival and in leukaemias in the form of unimpeded cell division of certain leucocyte types and of their precursors. Leucocytes behave like amoeboid cells and can be stimulated to migrate at random (chemokinesis) as well as directionally under the influence of a concentration gradient (chemotaxis) of distinct chemical agents (chemoattractants) in their environment. These two kinds of migration behavior are among the most sensitive criteria in studying the functional viability and intrinsic vital capacity of leucocytes.
There are various types of leucocytes: neutrophil, eosinophil and basophil phagocytes, mononuclear phagocytes (monocytes), and small mononuclear leucocytes (lymphocytes), as well as numerous different types of precursors. The lymphocytes in turn consist of different distinguishable subpopulations with various biological functions (T cells, B cells and the like). All types of leucocytes are formed in the bone marrow as multipotent parent cells capable of proliferation and differentiation. These parent cells are also the precursors of erythrocytes and thrombocytes. After maturation and passage into the blood stream, only the monocytes and lymphocytes retain their capacity to divide. The passage of the various kinds of leucocytes from the blood stream into the tissue as a result of chemoattraction in general is again associated with a further differentiation of the cells. For example, monocytes are converted into macrophages and basophil leucocytes into mast cells. Leucocytes are very sensitive cells, with a limited cell type-depending lifespan. The influence of external forces and the effect of numerous kinds of chemical substances or non-physiological conditions provoke both their functional and morphological damage.
So far, only small quantities of leucocytes (maximum of about 10.sup.10 cells per batch) can be obtained economically by existing preparation processes. For this purpose, for example, the entire leucocyte population is separated directly from the whole blood with the aid of so-called separating agents for two-phase systems by centrifugation or by gravitational forces [A. Boyum, Scand. J. Lab. Invest., 21, Suppl. 97 (1968)]. Similar processes are known to obtain only certain types of leucocytes, e.g. only neutrophil granulocytes of lymphocytes, by joint separation of all the other components of the blood. These known processes of direct separation are not suitable for obtaining larger quantities of leucocytes. [M. Graubner, et al, Inn. Med., 4, 316 and 380 (1977)]. They require large volumes of expensive separating agents for two-phase systems. Thus, a sample of 100 liters of blood would require about 150 liters of a "leucocyte two-phase-separation-fluid", for example, metrizoate-ficoll, density 1.077 g/ml, according to known procedures. [Nyegaard & Co., A/S Stjerne-Trykk, Nr. 746-75] The handling or in particular the centrifugation of very large volumes of whole blood or even larger volumes of dilutions of whole blood with such a separating agent remains an unsolved technical problem. The application of continuous flow-through centrifugation has been proposed as a solution to this problem. Attempts have also been made for magnetic separation of certain types of leucocytes, such as neutrophil granulocytes, which previously have phagocytosed additions of small magnetic iron particles. Techniques for separation of leucocytes from blood by aid of adsorption at specially prepared surfaces and filters (leucapheresis) are also in use. A further variant is the isolation of the so-called "buffy coat" in which leucocytes are partially aggregated or dead, by sedimentation at 1.times.g of all cellular blood constituents. For various reasons all these processes only allow preparation of leucocytes from smaller quantities of blood (maximum about 6 to 10 liters).
In addition to the mentioned difficulties in handling, extremely high costs of known processes for leucocyte separation result from the use of the necessarily large amounts of very expensive two-phase system separation and cell aggregation agents (metrizoate, dextran, and the like) [A. Boyum, Scand. J. Clin. Lab. Invest., 21, Suppl. 97 (1968)] and the specially prepared surfaces for differential leucocyte adsorption. In particular they deliver leucocytes whose functional and morphological integrity and viability are questionable. This applies above all to the continuous flow-through centrifugation method, in which the forces acting upon the leucocytes are highly irregular, and applies also to the isolation techniques by magnetic forces.
"Functional integrity" means substantially unimpaired random and directional locomotion capacity, unimpaired phagocytosis of foreign particles and cell-specific secretion of leucocyte products. "Morphological integrity" is commonly determined by the integrity of the cell shape and of the sub-cellular structures as well as by the negative adsorption of dyes of higher molecular weight by living cells in the dye exclusion test (vital staining).
A prerequisite for research of the biological functions of leucocytes, for the preparation of leucocyte hormones on a large scale, and for the medico-diagnostic and medico-technical applications of leucocytes is the availability of large quantities of functionally and morphologically intact and viable leucocytes. For example, in order to obtain about 1-10 mg of distinct leucocyte hormones, e.g., growth controlling factors, interferon, and the like, which are biologically active at nanomolar concentrations and are secreted by a molecularly homogeneous leucocyte culture, it is estimated that 50 kg of leucocytes must be available. This corresponds to processing about 10,000 liters of blood. Therefore, rapid, cell-sparing, effective and economical techniques are necessary to obtain pure leucocytes on a technical scale. The same considerations apply accordingly to thrombocytes.
It is, therefore, a primary object of this invention to provide a process for obtaining leucocytes from large amounts of blood.
It is another object of this invention to provide a process for obtaining leucocytes from blood in functionally and morphologically intact state.
It is another object of this invention to provide a process for obtaining high yields of large amounts of functionally and morphologically intact and viable leucocytes from blood.
It is still another object of this invention to provide a process which allows effective, economical, rapid and cellsparing preparation of a high yield of large amounts of leucocytes.
It is still another object of this invention to provide a process for obtaining from blood large amounts of thrombocytes which are functionally and morphologically intact and in a viable state.
These and other objects and advantages of the present invention will be evident from the following description of the invention.